I. Field of the Invention
The present invention relates generally to the fields of veterinary medicine, reproductive biology and diagnostics. More specifically, the present invention relates to methods and compositions for detecting early stage pregnancy.
II. Related Art
Pregnancy diagnosis allows for sound reproductive management in the dairy and beef industry. In general, artificial insemination is successful less than 50% of the time and the producer must either rely on overt signs of return to estrus (that are easily missed) or delay rebreeding until pregnancy failure is confirmed by one of the methods described above. Such delays are extremely costly and constitute a major economic loss to the industry.
An accurate pregnancy test for cattle which can be performed early and which has low false positives has long been sought. Several pregnancy tests are available, including a milk progesterone assay (Oltenacu et al., 1990; Markusfeld et al., 1990), estrone sulfate analysis (Holdsworth et al., 1982; Warnick et al., 1995), rectal palpation (Hatzidakis et al., 1993), ultrasound (Beal et al., 1992; Cameron and Malmo, 1993), and blood tests for pregnancy-specific antigens.
Each of these procedures has fallen short of expectations in terms of their practical, on-farm use. For example, measurements of milk or serum progesterone around day 18-22 yield unacceptably high rates of false positives (Oltenacu et al., 1990; Markusfeld et al., 1990). Rectal palpation can be used to detect pregnancy as early as day 35, but this procedure can lead to 5-10% or greater embryonic mortality (Oltenacu et al., 1990; Hatzidakis et al., 1993). Rectal palpation on day 50 causes less damage to the embryos, but has only marginal economic value because of its lateness (Oltenacu et al., 1990). Ultrasonography has an advantage over rectal palpation in accuracy, particularly before day 45 (Beal et al., 1992; Cameron and Malmo, 1993), but the instrument is expensive, its use requires considerable training, and there is a finite risk to the animal. A related procedure, Doppler sonography, is more accurate than rectal palpation (Cameron and Malmo, 1993), but again requires well trained personnel. The presence of estrone sulfate in urine or serum provides another test but is only useful after day 100 as concentrations rise (Holdsworth et al., 1982; Warnick et al., 1995).
The discovery of pregnancy-specific protein B (PSP-B) (Butler et al., 1982) provided a new approach to pregnancy diagnosis since it could be detected in the blood of pregnant cows by the fourth week of pregnancy (Sasser et al., 1986; Humblot et al., 1988). Others have developed immunoassays that may be based on an identical or immunologically similar antigen (Zoli et al., 1992a; Mialon et al., 1993; Mialon et al., 1994). In one case, the antigen (Mr ˜67 kDa) was called bovine pregnancy-associated glycoprotein (boPAG; now boPAG-1) (Zoli et al., 1992a); in the second, it was designated as pregnancy serum protein 60 (PSP60) (Mialon et al., 1993; Mialon et al., 1994). The immunoassays for PSP-B/boPAG1/PSP60 have certain disadvantages. First, positive diagnosis in the fourth week of pregnancy remains somewhat uncertain because antigen concentrations in blood are low and somewhat variable. Second, boPAG1 concentrations rise markedly at term (Sasser et al., 1986; Zoli et al., 1992a; Mialon et al., 1993) and, due to the long circulating half-life of the molecule (Kiracofe et al., 1993), the antigen can still be detected 80-100 day postpartum (Zoli et al., 1992a; Mialon et al., 1993; Mialon et al., 1994; Kiracofe et al., 1993), compromising pregnancy diagnosis in cows bred within the early postpartum period. Thus, the test can be carried out in dairy cows at day 30 only if artificial insemination (“AI”) is performed at or after 70 day post-partum.
Pregnancy-associated glycoproteins (PAGs) are structurally related to the pepsins. They are thought to be restricted to the hooved (ungulate) mammals and characterized by being expressed specifically in the outer epithelial cell layer (chorion/trophectoderm) of the placenta (Green et al., 2000; Hughes et al., 2003; Xie et al., 1997). At least some PAGs are catalytically inactive as proteinases, although each appears to possess a cleft capable of binding peptides (Guruprasad et al., 1996). It is estimated that cattle, sheep, and most probably all ruminant Artiodactyla possess dozens of PAG genes. The PAGs are highly diverse in sequence, with regions of hypervariability confined largely to surface-exposed loops.
Bovine pregnancy-associated glycoproteins (boPAGs/PSPB/PSP60) were discovered in attempts to develop pregnancy tests for livestock (Butler et al., 1982; Sasser et al., 1986; Zoli et al., 1991; Zoli et al., 1992a). In each attempt, rabbits were injected with extracts of placental cotyledons, and antibodies not directed against placental antigens were removed by adsorption with tissue extracts from non-pregnant animals. The resulting antisera provided the basis of an accurate pregnancy test for cattle and sheep as early as one month post-insemination.
Even in initial studies (Butler et al., 1982; Zoli et al., 1991; Xie et al., 1991; Xie et al., 1994; Xie et al., 1996), it was clear that the boPAGs were heterogeneous in molecular weight and charge, and as more isoforms have been purified it has become evident that they differ in their amino terminal sequences (Atkinson et al., 1993; Xie et al., 1997a). Further library screening has revealed additional transcripts in ruminants (Xie et al., 1994; Xie et al., 1995; Xie et al., 1997b) and the existence of PAGs in non-ruminant species such as the pig (Szafranska et al., 1995). PAG-like proteins (also known as ‘pepsinogen F’ or ‘pepsin F’) have been described in the horse and cat (Green et al., 1999; Guruprasad et al., 1996). Among the bovine PAGs that have been described are boPAG2, boPAG4, boPAG5, boPAG6, boPAG7, boPAG9, boPAG7v; boPAG9v; boPAG15; boPAG16; boPAG17; boPAG18; boPAG19; boPAG20 and boPAG21 (U.S. Pat. No. 6,869,770). Information regarding methods for diagnosing early pregnancy by assaying for such PAGs can be found, for example, in U.S. Pat. No. 6,869,770 and U.S. Patent App. Pub. No. 20050100975.
Most of the available tests for detecting pregnancy in cattle are less accurate prior to day 30 following breeding. Further, for many of the existing tests, skilled personnel are required. Thus, there is the need for an accurate and sensitive pregnancy test in cattle that can be performed quickly and easily prior to day 30 following breeding.